Objective 1. We have shown that the activation of CD4+T cells by combination of cytokines, for example, interleukin (IL-12) and IL-18, but not individual cytokine alone, turns on the CsA-resistant rapamycin-sensitive pathway. Since the intracellular target of rapamycin is mTOR, we have investigated the mechanism of activation of mTOR during T cell activation. Our data indicate that PKC-theta plays an important role in turning on mTOR pathway. We have shown that the translocation of PKC-theta to plasma membrane precedes the activation of mTOR downstream signaling molecules, p70S6K1 and AKT. Whereas migration of PKC-theta to the lipid-raft portion of the plasma membrane activates p70S6K1, the activation AKT requires the migration of PKC-theta to the plasma membrane. We are currently investigating the mechanisms involved in the activation of mTOR by PKC-theta. Objective 2. To investigate the physiological relevance of the mTOR pathway, we used the in vitro differentiation system of nave CD4+T cells, since the role of mTOR in T cell differentiation has been well documented. We were specifically interested in examining the role of p70S6K1 in in vitro differentiation of nave CD4+ T cells using CD4+ T cells from p70S6K1 mice. We have demonstrated that the lack of p70S6K1 greatly affected the in vitro differentiation of T helper (Th)-17 cells, whereas the development of Th1, Th2, and T regulatory cells were unaffected in the absence of p70S6K1. Furthermore, the major transcription factor in Th17 differentiation, retinoic acid receptor-related orphan receptor gamma T (RORT), remained unchanged. However, the acetylation of histone 3 at the promoter of IL17 was reduced in the absence of p70S6K1. In accordance with the in vitro data, the kinetics, but not the development, of experimental allergic encephalomyelitis (EAE) was affected with the loss of p70S6K1 expression. Collectively, our findings suggested that both in vitro and in vivo differentiation of Th17 were positively regulated by p70S6K1, and that other cytokines (e.g., IFN-) besides IL17 contribute the development of EAE.Currently, we are investigating the mechanisms involved in the role of p70S6K1 in the development of Th17 populations. Objective 3. The formation of reactive oxygen species (ROS) has long been associated with aging and the development of various pathophysiological conditions such as Alzheimer disease, atherosclerosis, and myocardial infarction, but recent studies have shown that ROS plays an important role in activating and regulating various signal transduction pathways in lymphocytes. We have observed that p70S6K1 plays an important role in sensing oxygen availability during activation of T cells. Using different culture conditions, we are able to demonstrate the differential production of ROS by wild type (WT) versus p70S6K1 knockout T cells. Since ROS is an important actor in the free radical theory of aging, and p70S6K1 has been shown to be a determinant of mammalian aging, we are interested in investigating the molecular mechanisms underlying the differential production of ROS versus mitochondrial respiration by WT versus p70S6K1 knockout T cells.